Within our continued effort aimed at developing conjugate vaccines for infectious diseases from carbohydrate antigens we have three ongoing projects. Two are concerned with a vaccine for cholera and one with a vaccine for anthrax. Existing vaccines for these diseases are based on cellular material and, in addition to having undesirable side effects, they do not provide long-term immunity. Development of vaccines for these two diseases is important from both the point of view of public health and of national interest. Development of a potent vaccine for cholera is important because of the involvement of our military in protecting US interests in developing third world countries. While anthrax does not constitute a major health problem in the civilized world, new concerns regarding anthrax have emerged because of potential use of some form of Bacillus anthracis, the etiological cause of anthrax, as a biological weapon. Our work towards a potent conjugate vaccine for cholera involves two approaches. In the first one, we synthesize oligosaccharides that mimic the structure of O-specific polysaccharide (O-PS) of Vibrio cholerae in the form suitable for conjugation, conjugate these antigens to suitable carriers, and serologically evaluate immunogenicity of the resulting neoglycoconjugates. In the second approach, we chemically modify polysaccharides isolated from bacterial pathogens to make them amenable for conjugation, conjugate the resulting synthons to suitable protein carriers, and use them as experimental vaccines. The approach towards a vaccine for anthrax is based on preparation of a neoglycoconjugate from a suitable carrier and the chemically synthesized tetrasaccharide side chain of the major glycoprotein of Bacillus anthracis exosporium. In the past, we have focused on improving diagnostic tools for the detection of presence of anthrax spores. Since preliminary work indicated that the anthrose-containing tetrasaccharide chain seemed to be highly specific for B. anthracis, during the period associated with this report we focused on testing the possibility of altering immunogenicity of the protective antigen (PA), which is protein in nature, by coupling it with the tetrasaccharide moiety from the BclA protein. Preliminary immunization studies suggested that this tetrasaccharide construct might enhance the immune response generated by PA. This was indicated by approximately 20% increase of protection of mice challenged with Ames spores. Unfortunately, due to lack of funds on the side of our collaborators, further work on optimizing the conjugates had to be temporarily discontinued. We are currently exploring possibilities to find new collaborations in this area. In the cholera project, while immunization studies are ongoing, we have focused on developing new methods that would allow localization of site of conjugation of synthetic antigens on the carrier protein by mass spectrometry. We have found that glycation sites in neoglycoglycoconjugates could be revealed, following proteolytic digest, by matrix-assisted laser desorption-ionization tandem mass spectrometry. Under these conditions, high energy collision-induced fragmentation occurs, resulting in spectra which reflect presence of fragments of the carbohydrate attached to amino acid sequences. As a result, we were able to identify in the past some conjugation sites on the carrier. More recently, we have refined the method by digesting a construct of the tetrasaccharide side chain of the Bacillus anthracis exosporium and the protein carrier bovine serum albumin. The glycoconjugate was digested with both trypsin and GluC V8 endoproteinases. Mass spectral analysis of fragments in the digest identified a higher number of glycopeptides than would be expected from a glycoconjugate composed of a carbohydrate-protein ratio of 5.4:1, which would have resulted in glycation occupancies of 18 specific sites. This discrepancy was due to the large number of glycoforms formed during conjugation of the synthetic carbohydrate. Knowing the glycation sites in neoglycoconjugate can be applied in quality control of conjugate vaccines. In addition, since the site of glycation may affect immunogenicity we expect the ability to determine the glycation sites to be helpful in optimizing the immunogenicity of future conjugate vaccines. One of the most important findings we have made in the past the discovery that bacterial O-SPcore antigens can be conjugated to proteins in the same, simple way as synthetic, linker-equipped carbohydrates by applying squaric acid chemistry. Introduction of spacers (linkers) to either O-SPcore antigens or protein carriers, which has always been involved in protocols for conjugation of these species, is not required. Conjugation of O-SPcore antigens to protein carriers by this method utilizes the free amino group that is inherent in the LPS core. It yields star-shaped, non-cross-linked, well defined neoglycoconjugates formed in the single-point mode of attachment of ligands to carriers. Compared to the previous approaches by other investigators, our conjugation method is much simpler and less laborious, and affords conjugates in higher yields and overall conjugation efficiency. Using O-PScore of Vibrio cholerae O1, serotype Inaba produced conjugates that were fully and specifically recognized by immune responses in humans recovering from infection. Such features are particularly attractive for development of conjugate vaccines for cholera. We are currently optimizing our laboratory-scale protocol to make it useful for preparation of large batches of experimental vaccine from O-PScore of Vibrio cholerae O1 and a recombinant tetanus toxin fragment cGMPs.